Figure 2. Inhibition of TBK1/IKKε exerts only minor effects on TNF-induced gene-activatory signalling.

(a-d) A549 WT cells were pre-incubated with either vehicle (DMSO) or MRT for 30 min, followed by stimulation with TNF (200 ng/mL) for the indicated times. (a) Lysates were analysed by western blotting. One representative experiment out of two is shown. * staining from previous p-JNK. Unprocessed original scans of blots are shown in Supplementary Figure 7 (b-d) Cells were then lysed, their total RNA extracted and RNA-Seq analysis performed. Samples from three independent experiments were obtaineded and analysed. (b) Principal-component analysis (PCA) of A549 samples based on transcriptome-wide expression level data is shown. (c) The heatmap illustrates the major change of expression across the dataset. The genes selected to be shown were the 100 most highly correlated with PC1 (see Fig 2b). For clarity of comparison the 'rlog' expression data of each row was zeroed at time-point 0 hr and then scaled by the standard deviation. The RNA-Seq raw dataset for b and c are available in the SRA repository and can be accessed by using the following BioProject accession: PRJNA422567 or SRA accession: SRP126844 (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP126844). (d) The Venn diagram represents the number of all transcripts significantly regulated upon 1 hr of TNF-stimulation in vehicle, MRT- or TPCA-1 -treated samples and the transcript overlap between those three groups. Corresponding transcripts can be found in supplementary table 3. Differential RNA-seq expression statistics (p-values) on contrasting biological triplicates, corresponding to samples obtained from three independent experiments (groups as in b-d) were estimated using DESeq2. Adjusted p-value statistics were calculated with the Benjamoni-Hochberg and IHW adjustment.